Transport of charged particles across cell membranes is mediated (and in substantial part, regulated) by membrane proteins (including those referred to as "voltage-sensitive" or "voltage-dependent" channels as well as "ligand-gated" channels).
In this discussion the terms "channel" and "channel protein" are used interchangeably without implying that a channel must necessarily consist of a single protein, although channels that have been isolated are believed to be single proteins.
Most channel proteins mediate the transport of one ionic species with substantially higher specificity than transport of other ionic species. It is common to name various channels after the ion for which they are specific: thus, there are sodium channels, potassium channels, calcium channels, etc.
In turn, ionic channels specific for the transport of one cation, may be further divided into various subcategories or channel types, based on the way they interact in response to electrical and/or chemical (pharmacological) stimuli. Calcium channels in particular, which have been identified in a number of different cell types, including neurons, appear to have differences (as well as similarities) in morphology, properties and/or function. Other such differences have not been directly related to cell types; in fact, different types of channels are normally present in the same cell.
Historically, neuronal calcium conductances were first divided into two categories based on the driving force that activates them: the high-threshold calcium conductance (HTCC) and the low-threshold calcium conductance (LTCC). In central neurons, HTCC is more prominent in the dendrites and LTCC is more prominent in the soma or cell body. Later, neuronal calcium channels were grouped into three categories: the T-channels, which are believed to be responsible for LTCC; the N-channels, the conductance properties of which showed imperfect correspondence to HTCC; and the L-channels which are not commonly represented in central neurons but of which the conductance properties also showed correspondence to HTCC.
These three categories can be further distinguished by differences in pharmacological properties. The L-channels are dihydropyridine-sensitive, i.e. they are effectively blocked by dihydropyridines, such as nifedipine and nitrendipene, whereas the T- and N-channels are dihydropyridine-resistant. To a large extent, some T-channels resist blockage by cadmium ions; more importantly, some T-channels are specifically blocked by alcohols (especially octanol) at 10.sup.-4 M or lower concentrations. Finally, the N-channels are said to be blocked by omega-conotoxin, a toxin isolated from the venom of the marine snail Conus geographicus, to which the L- and T-channels are resistant. Miller, R. J. Science, 1987, 235: 46-52.
However, the calcium channels responsible for the HTCC (both the calcium-dependent plateau potential component and the dendritic spike component of the HTCC) in Purkinje cells are activated at -50 mV, and are dihydropyridine-insensitive and also omega-conotoxin-resistant. These channels are specifically blocked by a low-molecular weight blocking agent isolated from the venom of funnel-web spiders. Llinas et all, Proc. Nat'l Acad. Sci. USA, 1989, 86:1689-1695. Furthermore, the dihydropyridine- and conotoxin-resistant calcium channels appear to be absent from inferior olivary and thalamic neurons. These calcium channels have been called P-channels because they were first described in Purkinje cells. Llinas, et all, Ann. N.Y. Acad. Sci., 1989, 560:103-111. P-type channels ave also been shown to exist in squid giant synapse and squid optic lobe.
Sodium and potassium channels are also of various types. See generally Hille, B. "Ionic Channels of Excitable Membranes," Sinauer Assoc. Inc., Sunderland, Mass., 1984.
Two main types of sodium channels are found in electrically excitable cells such as central neurons. One type of channel, responsible for the so-called "fast" sodium conductance, is specifically blocked by tetrodotoxin (TTX), a toxin isolated from puffer fish. A second type of sodium channel, responsible for the slow sodium current, is blocked by local anesthetic agents such as lidocaine. A distinctly different sodium channel is located in non-electrically excitable tissue such as epithelium, and is not blocked by TTX or the local anesthetics but is blocked by the diuretic amiloride.
The known blockers of K.sup.+ channels include tetraethylammonium (TEA), aminopyridines and quinine.
Agents that block calcium channels with high affinity and/or specificity as well as agents that activate calcium channels are useful as reagents in electrophysiological research. Availability of such agents is essential for understanding calcium channel properties and function. Such agents are especially useful in the design of prototype drugs and in drug screening.
The prior art calcium-channel blocking agents do not satisfy this need for one or more of the following reasons:
(a) Lack of Affinity for Particular Calcium Channels. The P-channel in particular is resistant to: omega-conotoxin; dihydropyridine and its derivatives; and alcohols. PA1 (b) Lack of Specificity. When developing substances that regulate a certain channel, specificity is a primary concern. PA1 (c) Unavailability in Large Amounts at Low Cost. It would be desirable to identify channel regulating agents which could be synthesized conveniently and at low cost. PA1 (d) Lack of Knowledge of Blocking Agent Mode of Action. The availability of blocking agents of different structure would help to elucidate the mechanism and to identify the site of blocking activity. This would in turn lead to design of improved blocking agents or of novel agents that had different calcium channel modulating properties, such as activation of calcium channels. PA1 (1) Alteration of the function of Na.sup.+ channels may be of therapeutic utility for the treatment of muscle spasms, torticollis, tremor, learning disorders, and Alzheimer's disease. Polyamines which block slow sodium channels would have additional utility as local anesthetic agents. Agents which act on the epithelian Na channel would be useful adjuncts in the treatment of cystic fibrosis and asthma and as antihypertensive agents. PA1 putrescine is said to cause an increase in cytosolic Ca.sup.2+ concentration in heart and kidney slices attributed to both an increase in extracellular Ca.sup.2+ influx and a simultaneous release of Ca.sup.2+ from mitochondria; PA1 spermine is said to inhibit spontaneous contraction of the uterus muscle in a manner than can be counteracted by increasing extracellular Ca.sup.2+ concentration; PA1 spermine and spermidine are said to have a relaxant effect on smooth muscles of the gut, uterus, respiratory tact and vasculature. PA1 to devise novel agents and methods for regulating (e.g. blocking, modulating or activating) ionic channels (including calcium, sodium, and potassium channels); PA1 to devise novel agents and methods for regulating transmitter release or synaptic transmission; PA1 to devise additional agents and methods for blocking ionic channels that overcome one or more of the disadvantages associated with known blocking agents described above; PA1 to use each of such regulating agents and methods to design prototypical drugs that regulate calcium, sodium, and/or potassium channels (with emphasis on drugs that block channels of central neurons); and to increase the understanding of ionic channel structure, properties and function; PA1 to design more active, less costly and/or otherwise improved substitutes for such agents and/or drugs; PA1 to design agents that have calcium, sodium, and/or potassium channel modulating functions other than activation or blockage. For example, agents which act on the G-protein in cell membranes alter the activity of ionic channels indirectly and so do agents that act on the NMDA receptor.
The calcium channel modulators of the present invention also have potential uses as prototypic drugs exhibiting anticonvulsant (e.g. anti-epileptic), anxiolytic, tranquilizing, anti-Alzheimer's, and/or memory-improving properties.
Analogous needs exist for substances that alter the ion-transport properties of other ionic channels, such as sodium or potassium channels, Such substances also find therapeutic uses such as:
(2) Drugs which modulate the activity of K.sup.+ channels would be useful as protective agents against the damaging effects of anoxic and ischemic disorders and hypertension, and would act to protect red blood cells against damage in malaria and sickle-cell disease.
Calcium blockers (as well as other types of calcium modulators), used separately, are expected to yield information about how the event of cell death is organized.
Chideckel, E. W., et al., Br. J. Pharmacol., 1986, 89:27-33 report that transient exposure to "polyamines" causes a relaxation of guinea-pig respiratory tract smooth muscle (trachealis muscle) in response to subsequent exposure to potassium ion extracellularly. This is contrary to the normal contractile response of this muscle exhibited in the absence of "polyamines" (or other calcium-ion entry blocking drugs such as nifedipine, verapamil, diltiazem or calcium ion-free solutions). The authors hypothesize inter alia that spermidine may have a Ca.sup.3+ antagonist function and that spermidine and certain other polyamines may have a calcium channel blocking activity, "perhaps" similar to that of the known Ca.sup.2+ -entry blocking drugs referred to above.
Spermidine was the only polyamine tried in the experiments described in this article, although effects of other polyamines related to membrane Ca.sup.2+ fluxes and cellular Ca.sup.2+ handling are cited from other literature as follows:
The authors do not mention the type of calcium channels that are said to be present in trachealis muscle (or for that matter in the other tissues mentioned). As to trachealis muscle, dihydropyridine sensitivity indicates that L-type channels might be implicated. As to the other tissues mentioned, no claim is made in this article that calcium channels were involved (many other factors or processes could influence the concentration of calcium in the cytosol). In fact, some of the results reported in this article are inconsistent with blockage of calcium channels, and (based on the disclosure of this article) cannot be attributed to activation of calcium channels because of the many other factors that may be at work, including actions on potassium channels or direct action on smooth muscle (papaverine-like effects).
Hirsh, S. R., et al, Psychopharmacology, 1987, 83:101-104 report that two polyamines, spermine and spermidine, administered intraperitoneally, at doses of 5-40 mg/kg, in rats caused a dose-dependent inhibition of spontaneous climbing and wheel running behavior, which the authors tentatively attributed to modulation by these polyamines of limbic dopamine function. Hirsch et al. report further that the mice remained alert during the period of reduced locomotor activity and appeared to be in good health.
Injected intracerebrally (in the striatum), these compounds failed to induce asymmetric behavior. On the other hand, these compounds did antagonize hyperactivity (when injected into the nucleus accumbens) which the authors (erroneously according to the present inventors) considered consistent with modulation of dopamine function but not dopamine receptor blockage. No direct or indirect effect on ionic (or specifically calcium) conductance is disclosed or suggested.
The effects reported erroneously attributed to dopamine by Hirsh et al. are hereafter referred to as "spermidine-like" effects.
Palade, P., J. Biol. Chem., 1987, 262:6149-6154 reports that the release of Ca.sup.2+ (pre-loaded into isolated subfractions of sarcoplasmic reticulum) usually observed upon addition of various release-inducing substances (including caffeine and thymol) cold be blocked by ruthenium red, certain organic polyamines (spermine, spermidine and triethylene tetramine) certain antibiotics (neomycin, kanamycin, tobramycin, gentamicin, streptomycin and clindamycin) and certain polypeptides (polylysine, polyarginine, some histones and protamine). The authors observed that these agents have only one feature in common: the presence of several amino groups. Based on the inability of the polyamines to affect calcium pump function, the authors concluded that the effect of ruthenium red, spermine, neomycin and polylysine (which have quite diverse structures) was not due to interference with the Ca.sup.2+ pump but to blockage of the sarcoplasmic reticulum calcium channels. Many of these agents appeared to block calcium release at (estimated) nanomolar concentrations. The authors also remarked that each compound's potency in inhibiting calcium release appeared to be related to the number of amine groups present in that compound: antibiotics appeared to be more potent on a molar concentration basis. However, since the experiments reported in this article measured only blockage of calcium release, they did not establish calcium channel involvement. Also, the reported results are limited to Ca.sup.2+ channels in the sarcoplasmic reticulum which have a large conductance (perhaps an order of magnitude greater than other known calcium channels) and, as stated by the authors, may be further limited to one type of Ca.sup.2+ channel in the sarcoplasmic reticulum. (The authors explicitly state that these compounds appear to be active on SR calcium channels that are insensitive to inositol triphosphate.). In general, SR calcium channels may be considered to be in a separate category not only because o their large conductance but also because of their location in the cell.
In a series of papers, Melchiorre, C., et al., report that certain polymethylene tetramines of the formula ##STR1## (wherein Ar is an aromatic group; R, R' are hydrogen or methyl and m,n are various integer combinations within the range 5-14) have M-2 muscarinic receptor blocking activity in guinea-pig heart atria and intestinal ileum. Several of these compounds are said to selectively block the atrial muscarinic receptor with considerably higher affinity than the ileal receptor, and thus could possibly serve to distinguish between the two receptors. Nothing is disclosed about calcium channels, but a general synthetic scheme for the non-aromatic moieties of the disclosed polyamines is provided: J. Med. Chem., 1989, 32:79-84; J. Med. Chem., 1985, 28:1643-1647; and Can. J. Physiol. Pharmacol., 1980, 58:1477-1483.
International Patent Application WO80/07098 discloses butyryl-tyrosinyl spermine and various analogs thereof which are said to be (potentially) useful for treating neurodisorders associated with an etiological agent binding to a glutamate receptor.